Life activities of organisms require proteins having various physiological activities. Many of these physiologically active proteins have a disulfide bond in their molecules. Also, they exist as oligomers such as dimers composed of homogenous or heterogeneous peptide chains and are biosynthesized in a state that a disulfide bond is formed between the peptide chains. While existence as a dimer is essential for many physiologically active proteins existing as a dimer to express their biological activity, proteins that are not necessarily required to exist as a dimer are also known.
As examples of proteins that are essentially required to be a dimer to express their physiological activity, a platelet-derived growth factor (PDGF) and so forth can be mentioned. Since each peptide chain of its dimer must be bonded to each peptide chain of a receptor, which is also a dimer, it is substantially essential for the protein to be a dimer to express its physiological activity (C. H. Heldin et al., Cell. Regul., 1, pp. 555-566, 1990).
However, it is also known that, in some physiologically active dimer proteins, one peptide chain (subunit) is considered to be responsible for an activity of binding to a receptor, enzymatic activity and so forth, while the other peptide chain is considered to be important for stability, solubility, biosynthesis process and so forth of the protein. That is, only a single subunit is essential for expression of substantial physiological activity. For example, although activin exists as a homodimer or a heterodimer in a living body, it has been reported that activin still has an activity of binding to its receptor and maintains about several percents of its biological activity even when it exists as a monomer (P. Husken-Hindi et al., J. Biol. Chem., 269, pp. 19380-19384, 1994). It has also been reported that prothoracicotropic hormone (PTTH), which exists in insects and has a physiological effect, exists in a living body as a homodimer, but about 50% of its biological activity is maintained in its monomer (J. Ishibashi et al., Biochemistry, 33, pp. 5912-5919, 1994). Further, although immunoglobulin G (IgG) is also a dimer, it is known that its antigen binding property is maintained in its monomer (G. M. Edelman, Biochemistry, 7, p. 1950, 1968).
As described above, some dimer proteins exhibiting physiological activities in a living body have their physiological activities even when they exist as a monomer. Further, monomers of some dimer proteins are bound to their receptors even though the proteins do not exhibit physiological activities when they exist as a monomer. Therefore, it is considered that, if a protein existing as a dimer is obtained as a monomer, the protein can be utilized as a protein having a physiological action or as a protein for inhibiting a physiological action of an original physiologically active protein by binding to its receptor to inhibit binding of the original physiologically active protein even though it does not have the physiological action.
Further, there are known many cases where proteins produced by different animal species exhibit a physiological activity in other animal species. For example, it is known that hirudin, which is a protein isolated from leech saliva, is bound to thrombin, which is a coagulation factor in blood, and thereby inhibits its protease activity. Proteins such as disintegrin and CHH-B that are bound to a platelet receptor and thereby inhibit platelet aggregation, are also known as proteins originating from snake venom. Among such heterogeneous proteins, CHH-B is a protein that is bound to glycoprotein Ib (GPIb), which is a glycoprotein on a platelet membrane, and thereby inhibits platelet aggregation. It has been reported that CHH-B is a heterodimer and its monomer also maintains its GPIb-binding activity and platelet aggregation inhibitory activity equally compared to those of the heterodimer (N. Fukuchi et al., WO 95/08573).
That is, it is considered that such a monomer of a physiologically active protein biosynthesized as a dimer as described above can be utilized as an action protein (agonist) or an action inhibitory protein (antagonist), and would be useful as a therapeutic agent for various diseases.
However, there are two major problems in obtaining a protein that is originally biosynthesized and exists as a dimer in a living body, in a form of a monomer in which its three-dimensional structure is maintained to such an extent that it should be stable and exhibit at least its biological activity.
The first problem is that preparation of a monomer is difficult. Two major methods are widely used to obtain a protein as a monomer that originally exists as a dimer. In one method, a dimer protein is partially reduced to cleave only a disulfide bond between subunits and then a newly generated free thiol groups are blocked. In the other method, a cysteine residue involved in a disulfide bond between subunits is replaced with an alanine residue or a serine residue by a technique of molecular biology and then a monomer is produced by using a protein synthesis system using an animal cell or the like. As for the aforementioned proteins, the former method is used for PTTH, CHH-B and IgG, while the latter method is used for activin and CHH-B. In the former method, since it is difficult to determine conditions for cleaving only the disulfide bond between subunits in some proteins, there have been few cases where a monomer was obtained while maintaining the activity. In the latter method, since the cysteine residue involved in the disulfide bond between subunits must be identified in advance and then a point mutation must be further inserted into the obtained gene. Therefore, it is a complicated method.
The second problem is that antigenicity may be expressed. When a protein existing as a dimer in a living body is obtained as a monomer, antigenicity may be exhibited because a region originally existing inside the molecule is exposed outside and may be recognized as a heterogeneous protein. In addition to this, in the first method for preparing a monomer, in which thiol groups are blocked after reduction, a compound used for blocking may exhibit antigenicity. Further, in the second method, in which a mutation is inserted, a partial structure containing the mutated amino acid may also exhibit antigenicity. Further, a protein originating from different animal species generally has antigenicity, and a protein obtained as a monomer also has a similar problem of antigenicity expression.
Meanwhile, it is well known that antigenicity of a protein can be decreased by polyethylene-glycolating (Bioconjugate Drugs, Drug Development, continued, Special Issue, Ed. by Inada and Tanimoto, Hirokawa Shoten, 1993; A. Abuchowski et al., J. Biol. Chem., 252, pp. 3578-3581, 1997). In particular, there have also been many reports on methods using polyethylene glycol having a functional group that is bound to a free thiol group of a cysteine residue in order to limit the number of polyethylene glycol molecules bound to a protein and their binding positions (G. N. Cox et al., WO98/32003; G. N. Cox et al., WO94/12219; R. J. Goodson, U.S. Pat. No. 5,206,344; L. G. Armes, WO92/16221). However, in all of these methods, a cysteine residue is artificially inserted in a part of a protein or replaced. There have been no reports that a useful protein is obtained by binding polyethylene glycol to a free thiol group without artificially replacing an amino acid in a subunit that originally forms a dimer.